With recent advances in testing techniques, the chemical, immunoserological and hematological examinations of blood have witnessed a remarkable mechanization so that it is by now certain that only if properly prepared samples were provided, such examinations could be carried through in short periods of time. For example, even in the outpatient setting, the doctor would be able to make a diagnosis based on blood examination data, thus contributing much to the diagnosis and therapy of diseases.
As to the pretreatment for hematological examinations using whole blood as a sample, mere admixing of the blood with an anticoagulant, which is not time-consuming, is sufficient so that the sample can be almost immediately set in an analyzer.
However, in biochemical or immunoserological examinations using the serum fraction of blood, it is necessary to coagulate the blood once and, then, separate the serum by centrifugation or the like and the procedure is rather time-consuming. Therefore, in order to reduce the time required for the whole examination procedure from the pretreatment of a sample to the output of test data, mere shortening of the analysis time by mechanization of the analytical procedure is insufficient and it is necessary to shorten the time required for separation of serum.
Meanwhile, glassware has heretofore been used as the blood examination vessel for accommodating the blood to be tested, allowing it to coagulate therein, and separating the serum by centrifugation. However, glassware is vulnerable to mechanical impact and, when it is broken, the test sample that issues out or splashes may cause the examiner to be infected by pathogenic bacteria and, as an additional problem, the necessary blood sampling for reexamination adds to a burden on the patient. For these reasons, plastic vessels have come into popular use in recent years. However, such plastic ware has been found disadvantageous in that the formed elements of the blood (hereinafter referred to as blood components) such as platelets, various blood proteins, and especially the fibrins which are formed in the final stage of the blood coagulation process, are very liable to deposit on the inside wall of the plastic ware and thereby exert untoward effects on examination results. Moreover, as will be described in detail hereinafter, it is common practice to employ a mineral substance or an organic substance, such as ellagic acid, as a blood coagulation accelerator in blood examination ware but such blood coagulation accelerator tends to encourage deposition of said blood components on the vessel wall and the blood components once deposited will not easily be detached from the inside wall of the vessel under the routine conditions of centrifugation such as about 1000 to 1800 G.times.5 minutes. As a result, owing to the high shear force of centrifugation acting on the interface between the inside wall and the clot, the platelets and red blood cells are destroyed and their contents leak out to affect the examination results.
To overcome these disadvantages, Japanese Kokai Publication Sho-58-105063 and Japanese Kokai Publication Sho-58-105064 proposed a method which comprises disposing a blood coagulation accelerator and a nonionic surfactant concomitantly on the inside wall of the ware, for instance. However, with the recent rapid development of high sensitivity techniques in the field of immunoserological examination, analogues of nonionic surfactants are being used as sensitizers on more and more occasions. If the test serum is contaminated with a nonionic surfactant, oversensitizing reactions occur in immunoserologic parameters to present the problem of inaccuracy leading to false positive tests.
On the other hand, for efficient separation of plasma from the blood to be analyzed, there is a protocol involving addition of a blood anticoagulant such as an ethylenediaminetetraacetic acid salt or a citrate to the blood sample. In hematological examinations using an anticoagulant, too, the deposition of blood components, particularly platelets, on the plastic surface may be a cause of trouble, although the frequency of the trouble is not high. If platelets stick to the inside wall of blood test ware, the platelet count may show on abnormally low value or confound blood coagulation function values. Moreover, where an emergency chemical examination is required, it is common practice to use a heparin salt which is a kind of anticoagulant but if the deposition of platelets occurs in such cases, various enzymes of platelet origin leak out into the plasma with time so that the related examination parameters tend to show abnormally high values. These events are less frequent as compared with the coagulation of blood and have so far attracted little attention but are now pointed out as a serious problem as an omnibus, accurate and rapid blood examination is demanded.
The above problem of deposition of blood components has been pointed out with reference to plastic blood test ware but recently the adverse influences of the deposition and activation of platelets on examination results have been pointed out for blood examination glassware as well and improvements are being sought just as for plastic ware.
Since plastic ware for blood examination is intrinsically low in the potential to activate blood coagulation XII factor and XI factor, it takes by far a longer time for the blood to coagulate in plastic ware than in glassware and, therefore, plastic ware has so far been of low practical value.
Therefore, attempts have been made to shorten the blood coagulation time by coating the inside wall of blood test ware with a finely divided mineral substance such as glass, kaolin, bentonite, silica, cerite, or the like or a blood coagulation accelerator such as ellagic acid as taught in Japanese Kokai Publication Sho-58-195151 or accommodating in the ware a substantially blood-insoluble and chemically inert nonwoven cloth or plastic sheet matrix on which said finely divided particles have been immobilized as taught in Japanese Kokai Publication Sho-58-105064.
When a blood coagulation-accelerating substance is to be coated on the inside wall of a blood test ware or immobilized on a carrier, a suspension of finely divided particles of such substance either in pure water or in a mixture of alcohol and pure water is prepared and spray-coated on the inner surface of the ware or a carrier material is dipped in such a suspension, dried, cut to size, and accommodated within the blood test ware.
However, such a treating suspension is susceptible to the attack of microorganisms and unless it is properly handled, may cause contamination of a blood sample with microorganisms. Furthermore, when a water-soluble macromolecular compound such as polyvinylpyrrolidone or a modified cellulose is incorporated in said suspension as a binder for said coagulation accelerator powder or a viscosity adjusting agent for the suspension as is generally practiced, the water-soluble macromolecular compound serves as a good nutrient source for microorganisms so that the above-mentioned tendency of the treating suspension to be a microbial contamination risk factor is further encouraged.
As the proliferation of microorganisms progresses, condensation products will be accumulated in the treating suspension to cause troubles such as clogging of the spray nozzle, marked loss of coated surface evenness, and biases in the density of particles immobilized on the carrier in the dipping stage, all of which add up to measurement errors. The risk of microbial contamination is not confined to the current risk associated with degradation of the treating suspension but is a persistent drawback for the shelf-life of the ware unless the method for storage of the ware is wholesome.
Therefore, unless manufactured in a sterile environment, a blood test ware containing a blood coagulation accelerator may have to be sterilized with actinic radiation such as gamma-rays, electron beams, etc. or a chemically reactive gas such as ethylene oxide gas. In any of such procedures the radiation dose or the concentration of the reactive gas, heating temperature, exposure time and other sterilizing conditions must be adjusted according to the contamination status prior to sterilization and, thus, very delicate control is required.
Moreover, where the sterilizing load has been severely contaminated, rugged sterilizing conditions are required so that the load may sustain irreversible modification, deformation, and other damages. Moreover, in the storage after sterilization, the sterility once established will be jeopardized unless the ware is properly packaged.
One of the possible effective approaches to solving the above problems is to impart antimicrobial activity to the very coagulation accelerator. By such a technique, the above-mentioned microbial contamination would be inhibited and even if sterilization be needed, mild sterilizing conditions would be sufficient, with the result that the physical and chemical changes of the load due to sterilization could be prevented or suppressed. Furthermore, the packaging of the blood test ware could be simplified.
As regards antibacterial and antifungal agents which are generally used for prevention of microbial contamination, a large number of compounds inclusive of those for food use are already known and in use. However, the large majority of these antibacterial and antifungal agents are water-soluble and, therefore, if the blood is drawn into a blood test ware in which a blood anticoagulant supplemented with such an antimicrobial agent has been accommodated, the antimicrobial agent may dissolve out into the blood to confound various chemical tests. Moreover, when the antibacterial or antifungal agent is a water-soluble heavy metal salt, it modifies the enzymes associated with blood coagulation and the resulting deactivation of the enzymes prevent coagulation of the blood and make it difficult to achieve the objective such as separation of serum.
Where the specimen to be analyzed is plasma, it is a routine procedure to mix the blood with an anticoagulant and centrifuge the mixture to separate plasma from the solid fraction. Generally speaking, in order to avoid contamination of plasma with substances liberated from formed elements of the blood and other matter and the consequent interference with tests, the plasma obtained by centrifuging blood in the above manner is transferred to a different container and stored. Recently, however, for the purpose of protecting the examiner against infection via the patient's blood, a procedure which does not require a transfer to another ware is demanded. Therefore, the use of a plasma separator comprising a thixotropic fluid as disclosed in Japanese Kokai Publication Hei-2-168159 or the use of a separator for provision of a partition between the plasma layer and the solid component layer as taught in Japanese Kokai Publication Hei-5-26873 has been recently employed.
However, the inside surface of the plastic ware is hydrophobic and the blood cells and proteins are adsorbed thereon as mentioned above. Particularly platelets are adsorbed with a high affinity and because LDH (lactic dehydrogenase), CPK (creatine kinase), K (potassium), etc. occur at higher levels in platelets than in plasma, these components are released from the platelets adsorbed on the plastic surface and it is inevitable that the values of these test parameters are considerably affected.
Therefore, even if the above-mentioned procedure of providing a partition between the plasma layer and the solid component layer is followed, the gradual release of enzymes and others from the blood cells adsorbed on the inside wall of the ware containing the plasma is unavoidable and interferes with tests. These adverse effects are particularly remarkable when the plasma is stored in the refrigerator for reexamination.
Having overcome the above disadvantages of the prior art, the present invention has for its primary object to provide a blood component deposition-preventing agent which is capable of inhibiting deposition of blood components effectively without causing the problem of false positive reactions in the immunoserological tests.
The second object of the present invention is to provide a blood coagulation accelerator comprising an antibacterial composition which has its own blood coagulation-accelerating activity and yet substantially does not interfere with blood coagulation activity or confound serum biochemical tests.
The third object of the present invention is to provide a plastic ware and a matrix for blood examination which do not influence test values owing to release of substances from blood cells even when used in tests on plasma.